Lipidated immune response modifier compound compositions, formulations, and methods

ABSTRACT

The compound N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide is a useful drug compound for enhancing immune response and can be used, for example, as a vaccine adjuvant and a cancer treatment.

BACKGROUND

There has been an effort in recent years, with significant success, todiscover new drug compounds that act by stimulating certain key aspectsof the immune system, as well as by suppressing certain other aspects(see, e.g., U.S. Pat. No. 6,039,969 (Tomai et al.) and U.S. Pat. No.6,200,592 (Tomai et al.). These compounds, referred to herein as immuneresponse modifiers (IRMs), appear to act through basic immune systemmechanisms known as Toll-like receptors (TLRs) to induce selectedcytokine biosynthesis, induction of co-stimulatory molecules, andincreased antigen-presenting capacity.

Many IRMs may be useful for treating a wide variety of diseases andconditions. For example, certain IRMs may be useful for treating viraldiseases (e.g., human papilloma virus, hepatitis, herpes), neoplasias(e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis,melanoma), T_(H)2-mediated diseases (e.g., asthma, allergic rhinitis,atopic dermatitis), and auto-immune diseases. Certain IRMs may also beuseful, for example, as vaccine adjuvants.

Many known IRMs are imidazoquinoline amine derivatives (see, e.g., U.S.Pat. No. 4,689,338 (Gerster)), but other compound classes are known aswell (see, e.g., U.S. Pat. No. 5,446,153 (Lindstrom et al.); U.S. Pat.No. 6,194,425 (Gerster et al.); and U.S. Pat. No. 6,110,929 (Gerster etal.); and International Publication Number WO2005/079195 (Hays et al.).

In view of the great therapeutic potential for IRMs in the treatment ofa wide variety of diseases and conditions, and despite the importantwork that has already been done, new compounds that can effectivelymodulate the immune response, by induction of cytokine biosynthesis orother mechanisms, are still needed.

SUMMARY

The present invention provides, in one aspect, a new compound useful forinducing cytokine biosynthesis. The compound (i.e.,N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide)has the following formula (I):

Pharmaceutically acceptable salts of the compound may also be used.

The compound of Formula I has unexpectedly beneficial properties interms of biologic activity. It is particularly desirable forincorporation into liposome based formulations. It appears that suchformulations are surprisingly effective at boosting localized immuneresponse with reduced systemic TNF induction.

The ability to induce cytokine biosynthesis in animals makes thecompound of Formula I useful for treating a variety of conditions suchas viral diseases and tumors that are responsive to such changes in theimmune response. Accordingly, the present invention further providesmethods of inducing cytokine biosynthesis in an animal, treating a viralinfection and/or treating a neoplastic disease in an animal byadministering an effective amount of a compound of Formula I to theanimal. The present invention further provides a method of vaccinatingan animal comprising administering an effective amount of a compound ofFormula I to the animal as a vaccine adjuvant.

The invention further provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of a compound of Formula I. In some embodiments, thepharmaceutical composition further comprises an antigen (e.g., avaccine). In some embodiments of the pharmaceutical composition, thecompound of Formula I is incorporated in a homogeneously dispersedformulation. In some embodiments of the pharmaceutical composition, thecompound of Formula I is incorporated in an emulsified formulation. Insome embodiments of the pharmaceutical composition, the compound ofFormula I is incorporated in an oil-in-water formulation (for exampleformulations comprising soybean oil, TWEEN 80, SPAN 85, and PBS). Insome embodiments of the pharmaceutical composition, the compound ofFormula I is incorporated into a liposome-based formulation.

Used as a vaccine adjuvant to an antigen vaccine, the compound ofFormula I increases the antibody response to the vaccine. It candecrease the amount of antigen vaccine required to achieve adesired\therapeutically effective antibody response. For example, it canreduce the amount of vaccine antigen needed by 2-fold, 10-fold, 15-fold,25-fold, 50-fold, or as much as 100-fold or more.

As illustrated in part by the non-limiting examples set forth herein,the compound of Formula I is useful for a wide range purposes, includingbut not limited to such things as a vaccine adjuvant for influenzavaccines. For example, when used as a vaccine adjuvant, the compound ofFormula I in combination with an influenza vaccine antigen providesprotection for H1N1 influenza infection (as well as influenza A, B, andswine flu). In particular, when used as a vaccine adjuvant, the compoundof Formula I in combination with hemagglutinin antigens providesprotection for H1N1 influenza infection.

The compound of Formula I induces cytokine production primarily at thesite of administration (or at a local site of application) and can do sowithout substantial systemic cytokine induction, which may be importantfor reducing side effects. For example, the compound of Formula I caninduce TNF production primarily at the site of administration (or at alocal site of application) without inducing systemic TNF levels abovethe background level (i.e. the level measured systemically prior toadministration of the compound of Formula I). In some applicationssubcutaneous injection of the compound of Formula I can be used toinduce cytokine production (such as TNF production) in the localdraining lymph nodes, but not peripheral lymph nodes. For example,subcutaneous injection of the compound of Formula I can induce cytokineproduction (such as TNF production) in the local draining lymph nodes atlevels at least 2 times, 3 times, 5 times, 10 times, or as much as 100times greater or more than in the peripheral lymph nodes

In addition to the compound of Formula I, it is believed that thecompoundN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)hexadecanamidemay be synthesized using a similar synthetic route and may also be usedfor the same uses, pharmaceutical compositions, and formulations as thecompound of Formula one set forth herein.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a”, “an”, “the”, “at least one”, and “one or more” areused interchangeably.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

“Ameliorate” refers to any reduction in the extent, severity, frequency,and/or likelihood of a symptom or clinical sign characteristic of aparticular condition.

“Antigen” refers to any substance that may be bound by an antibody in amanner that is immunospecific to some degree.

“Induce” and variations thereof refer to any measurable increase incellular activity. For example, induction of an immune response mayinclude, for example, an increase in the production of a cytokine,activation, proliferation, or maturation of a population of immunecells, and/or other indicator of increased immune function.

“Liposome” or “liposome based” as used herein refers generally to aself-assembling particle composed of amphipathic molecules such as, butnot limited to lipid, lipid-like, or polymeric substances. They can alsoinclude lipopeptides and glycolipids.

“Symptom” refers to any subjective evidence of disease or of a patient'scondition.

“Therapeutic” and variations thereof refer to a treatment thatameliorates one or more existing symptoms or clinical signs associatedwith a condition.

“Treat” or variations thereof refer to reducing, limiting progression,ameliorating, preventing, or resolving, to any extent, the symptoms orsigns related to a condition.

The compoundN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamidedescribed herein may be in any of its pharmaceutically acceptable formsincluding solid, semi-solid, solvate (e.g., hydrate), wholly orpartially dissolved (e.g., in a pharmaceutical composition), ordispersed in a pharmaceutically acceptable carrier. It will also beunderstood that any pharmaceutically acceptable salt form of thecompound of Formula I(N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide)may also be used.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the description,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

DETAILED DESCRIPTION Example 1 N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide

A solution of valeric anhydride (6.03 g) and pyridine hydrochloride(0.198 g) in pyridine (8.28 g) was added to a solution of3-amino-4-chloroquinoline (2.94 g) in pyridine (5.0 g) and the reactionwas stirred at room temperature for 16 hours followed by heating at 60°C. for 3 hours. The reaction was concentrated under reduced pressure andsodium carbonate (15 mL of a 10% aqueous solution) was added. Thereaction was stirred for 30 minutes and then filtered. The resultingsolid was washed with water (60 mL) and dried under vacuum for 4 hoursto provide 4.59 g of crude N-(4-chloroquinolin-3-yl)valeramide as brownflakes. The crude product was recrystallized from heptane (10 mL) andthe recovered product was further purified by soxhlet extraction usingrefluxing heptane for 16 hours. The collection flask from the soxhletextraction apparatus was cooled in a freezer for 2 hours. The resultingsolid was collected by filtration and dried under vacuum to yield 2.00 gof N-(4-chloroquinolin-3-yl)valeramide as a white solid.

Part B

A solution of 4-amino-1-butanol (7.68 g) and pyridine (7.00 g) indichloromethane (100 mL) was chilled in an ice bath and a solution ofbenzylchloroformate (14.37 g) in dichloromethane (100 mL) was slowlyadded with stirring over a period of thirty minutes. The ice bath wasremoved and the reaction was stirred for an additional 16 hours.Hydrochloric acid (1.2 M, 200 mL) was added and phases were separated.The organic phase was dried (MgSO₄), filtered and concentrated underreduced pressure. The resulting residue was recrystallized from tolueneand dried under vacuum to provide 5.15 g of benzyl(4-hydroxybutyl)carbamate.

A solution of N-hydroxyphthalimide (3.36 g), benzyl(4-hydroxybutyl)carbamate (4.18 g) and triphenylphosphine (7.41 g) indichloromethane (100 mL) was chilled in an ice bath and approximatelytwo-thirds of a solution of diisopropylazodicarboxylate (DIAD, 5.68 g)in dichloromethane (50 mL) was slowly added with stirring. The internaltemperature of the reaction was monitored and the addition of the DIADsolution was stopped when an exotherm could no longer be detected. Theice bath was removed and the reaction was allowed to warm to roomtemperature. The reaction was concentrated under reduced pressure andthe resulting residue was dissolved in ethanol (200 proof, 100 mL).Hydrazine (1.98 g, 35% in water) was added and the reaction was stirredfor 6 hours. The reaction was cooled in the freezer and the resultingsolid was removed by filtration. The solid was washed with ethanol (50mL). The combined filtrate was concentrated under reduced pressure anddiethyl ether (100 mL) was added. Insoluble impurities were removed byfiltration and 2.0 M HCl in ether (10 mL) was added to the solution. Aprecipitate formed immediately. The crude product was added to toluene(100 mL) and heated at reflux temperature for one hour. After cooling toroom temperature, the solid product was recovered by filtration, washedwith toluene, and dried under vacuum to yield 3.76 g of benzyl(4-aminooxybutyl)carbamate.

Part C

N-(4-chloroquinolin-3-yl)valeramide (1.97 g), benzyl(4-aminooxybutyl)carbamate (2.99 g), triethylamine (0.89 g) and2-propanol (40.69 g) were combined and heated at 80° C. for 3.5 hours.The reaction was cooled to room temperature, filtered, and the filtrateconcentrated under reduced pressure. Dichloromethane (20 mL) was addedto the resulting solid and the mixture was stirred for twenty minutes.Undissolved solid was removed by filtration and the filtrate was washedwith two 10 mL portions of water that had been made slightly acidic bythe addition of 20 drops of hydrochloric acid (1.2 M). The organicfraction was dried and concentrated under reduced pressure. The crudesolid was recrystallized from tetrahydrofuran to provide 2.56 g ofbenzyl 4-{[2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butylcarbamate.

Part D

Benzyl 4-{[2-butyl-1H-imidazo [4,5-c]quinolin-1-yl]oxy}butylcarbamatehydrochloride (10.05 g) was dissolved in dichloromethane (80 mL) andextracted with a solution of sodium carbonate (2.02 g) in 30 ml H₂O. Theorganic layer was cooled in an ice bath and a solution ofm-chloroperbenzoic acid (5.93 g, 1.24 eq) dissolved in dichloromethane(30 mL) was slowly added. After 6 hr, ammonium hydroxide (10 mL of a28-30% aqueous solution) was added to the reaction. A solution ofbenzenesulfonyl chloride (6.96 g) dissolved in 10 ml dichloromethane wasslowly added with vigorous stirring. The cooling bath was removed andthe reaction was stirred for an additional 12 hours. The reaction wasdiluted with water (100 mL) and the organic and aqueous fractions wereseparated. The aqueous fraction was extracted with dichloromethane (30mL). The combined organic fractions were washed with two 90 ml portionsof 5% sodium carbonate.

The dichloromethane solution was transferred to a distillation apparatusand 1-pentanol (50 mL) was added. This was warmed to 40° C. and thedichoromethane was removed under reduced pressure. Concentratedhydrochloric acid (50 ml) was then added and the reaction was stirredand heated to 80°. After 11 hours, the solution was cooled to roomtemperature and diluted with water (100 mL). The aqueous fraction wasseparated from the 1-pentanol and the 1-pentanol was extracted withwater (25 mL). The aqueous fractions were combined. 1-Pentanol (50 mL)was added to the combined aqueous fraction and this was cooled in anice-bath. With vigorous stirring, solid sodium carbonate was added tobring the pH to 9-10. The mixture was transferred to a separatory funneland the fractions were separated. The aqueous fraction was extractedwith two 25 ml portions of 1-pentanol. The combined 1-pentanol fractionswere dried over sodium sulfate and filtered to provide1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine dissolved in1-pentanol.

The maleate salt of1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine was preparedby dissolving maleic acid (4.83 g) in 1-pentanol (50 mL) and adding itwith stirring to the solution of1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine in1-pentanol. The resulting precipitate was collected by filtration anddried to yield 7.69 g of1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine bis maleatesalt. ¹H-NMR (DMSO-d6): δ 0.96 (t, 3H), 1.44 (m, 2H), 1.7-1.95 (m, 4H),2.02 (m, 2H), 2.8-3.1 (m, 4H), δ 4.43 (t, 2H), 6.07 (s, 4H), 7.57 (t,1H), 7.73 (t, 1H), 7.80 (d, 1H), 8.16 (d, 1H). Broad peaks for theammonium protons are seen at approximately δ 7.8 and δ 8.7.

As an alternative the fumarate salt of1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine was preparedby the following procedure.1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (6.53 g) wasdissolved in 2-propanol (75 mL) and decolorizing carbon was added. Thereaction was heated to reflux, filtered while hot, and cooled to roomtemperature. A solution of fumaric acid (2.5 g) in 2-propanol was addedand the reaction was heated at reflux temperature for 5 minutes. Uponcooling to room temperature a precipitate formed. Filtration followed bydrying the product under vacuum yielded 6.6 g of1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine fumarate.¹H-NMR (DMSO-d6): δ 0.95 (t, 3H), 1.42 (m, 2H), 1.70-1.92 (m, 4H),1.92-2.10 (m, 2H), 2.85-3.05 (m, 4H), 4.34 (t, 3H), δ 6.46 (s, 2H), 7.30(t, 1H), 7.47 (t, 1H), 7.60 (d, 1H), 8.02 (d, 1H). A broad ammonium peakappears at δ 6.77.

Part E

1-(4-Aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine fumarate(1.30 g) was dissolved in dichloromethane (25 mL) and the solutionwashed with 3×15 ml portions of saturated sodium carbonate. The organicfraction was then washed with 15 ml saturated sodium chloride and driedover MgSO₄. The solution was filtered, the solvent removed under reducedpressure and the product was dried under vacuum to give 0.79 g of1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine as the freebase.

The 1-(4-aminobutoxy)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine wasdissolved in dichloromethane (20 mL) and methanol (5 mL). Stearic acid(0.71 g) was added and the reaction was stirred to dissolve the stearicacid. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC, 0.45 g)was added and the reaction was stirred at ambient temperature for 16hours. An additional portion of EDC was added (0.23 g) and the reactionwas stirred for an additional 24 hours. Final portions of stearic acid(0.22 g) and EDC (0.37 g) were added to drive the reaction to completionand the reaction was stirred at ambient temperature for another 24hours. The reaction was concentrated under reduced pressure and theresulting residue was purified by flash column chromatography using aBiotage chromatography system (Si40+M2358-1 SiGel column, 85:15dichloromethane/methanol isocratic elution). The semi-pure product waspurified by flash column chromatography two more times using a 90:10dichloromethane/methanol isocratic elution, followed by a 95:5dichloromethane/methanol isocratic elution The fractions containingproduct were concentrated to yield 1.12 g ofN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamideas an off white waxy solid.

¹H-NMR (CDCl₃): δ 0.89 (t, 3H), 1.01 (t, 3H), 1.14-1.42 (m, 28H), 1.50(m, 2H), 1.65 (m, 2H), 1.74-1.94 (m, 4H), 2.02 (m, 2H), 2.20 (t, 2H),2.95 (t, 2H), 3.40 (q, 2H), 4.33 (t, 2H), 5.59 (t, 1H), 6.10 (broad s,2H), 7.39 (m, 1H), δ 7.57 (m, 1H), 7.83 (d, 1H), 8.07 (m, 1H).

Example 2

The vaccine adjuvant activity of N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide (Cmpd of Example 1) wasevaluated in mice immunized with recombinant hemagglutinin 1 (HA). IgG2aantigen specific antibody response was measured using five differentpreparations (1. HA alone (control); 2. HA+resiquimod (comparatorpreparation); 3. HA+Cmpd of Example 1 formulated indioleoylphosphatidylcholine (DOPC) (liposome formulation); 3. HA+Cmpd ofExample 1; 5. HA+DOPC (control).

The Cmpd of Example 1 and resiquimod were individually prepared asaqueous suspensions in phosphate buffered saline (PBS). The Cmpd ofExample 1 formulated in DOPC liposome formulation was prepared asfollows. A stock solution of the Cmpd of Example 1 was prepared inchloroform at a concentration of 10 mg/ml. A stock solution ofdioleoylphosphatidylcholine (DOPC) was also prepared in chloroform at aconcentration of 10 mg/ml. Aliquots of each stock solution were combinedto provide a solution containing DOPC and the Cmpd of Example 1 at amass ratio of 10:1, respectively. The solution was blown to dryness andresuspended in sterile PBS by probe sonication.

Groups of 5 mice each were immunized subcutaneously with 10 μg of HAantigen in PBS, alone or in combination with 1 mg/Kg of the compoundscited in Table 1. DOPC control animals received the same amount of DOPCas that prepared with the Cmpd of Example 1. The mice were boosted withthe same combinations 2 weeks and 4 weeks following the initialimmunization. At 7 weeks post immunization, the mice were bled and theHA-specific IgG2a titers were determined. This determination wasperformed by serial dilution of the serum samples by standard serumELISA in HA-coated microtiter plates. IgG2a data is presented as theserum dilution achieving the end point (2× baseline) and is thegeometric mean for the 5 mice per group.

TABLE 1 HA Specific IgG2a, Serum Dilution End In Vivo Immunization GroupPoint HA 3.30E+03 HA + Resiquimod 1.00E+05 HA + Cmpd of Example 1/DOPC3.30E+06 HA + Cmpd of Example 1 1.42E+04 HA + DOPC 5.00E+03

Example 3

Antigen dependent interferon-gamma (IFNgamma) responses were determinedin spleenocyte cultures established from the same animals for whichIgG2a antibody responses were determined in Example 2. The spleens fromthe animals were removed, combined to form two pools for each group of 5animals, minced to create single cell suspensions, and placed in culturein 96 well microtiter plates. Each pool generated three wells for acontrol PBS challenge and three wells for a 10 mg HA challenge. Thecultures were then incubated at 37° C. for 72 hours. The medium was thenremoved and the interferon-gamma generated was measured (pg/ml) by anELISA assay (Table 2). The IFNgamma data is reported as the geometricmean value for each pool using triplicate measurements.

TABLE 2 In Vitro Challenge of Isolated Spleenocytes, (IFNgamma, pg/ml)Control PBS HA Antigen In Vivo Immunization Group Challenge Challenge HA4.32 157.87 HA + Resiquimod 3.84 91.88 HA + Cmpd of Example 1/DOPC 5.841808.19 HA + Cmpd of Example 1 4.82 293.51 HA + DOPC 1.7 231.97

Example 4

The effect of N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide (Cmpd of Example 1) toinduce the formation of systemic tumor necrosis factor (TNF) in vivo wasevaluated in mice. Systemic TNF induction was measured using fourdifferent preparations (1. PBS (control); 2. resiquimod (comparatorpreparation); 3. resiquimod formulated in dioleoylphosphatidylcholine(DOPC) (comparator preparation); 4. Cmpd of Example 1 formulated indioleoylphosphatidylcholine (DOPC) (liposomes).

Compound of Example 1 formulated in dioleoylphosphatidylcholine (DOPC)liposomes was prepared as described in Example 2. Resiquimod formulatedin DOPC was prepared in an analogous manner to the Cmpd of Example 1 inDOPC. The resiquimod preparation was made as an aqueous suspension inPBS.

Mice were injected subcutaneously with preparations containing 1 mg/Kgof each test compound (i.e. resiquimod or Cmpd of Example 1). At onehour and at three hours post dose, the mice were bled and systemic TNFwas measured in the serum (pg/mL) by ELISA assay. The results arepresented as the geometric means obtained for each group of fiveanimals. The data in Table 3 shows that subcutaneous injection ofresiquimod in various formulations induces a systemic TNF response,whileN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide(Cmpd of Example 1) does not induce a systemic TNF response. This can beimportant in providing localized immune system enhancement withoutsystemic TNF side effects.

TABLE 3 TNF concentration (pg/mL) at Times Following Treatment Treatment1 hour 3 hour PBS <5 <5 Resiquimod 1140.41 <5 Resiquimod/DOPC 647.67 <5Cmpd of Example 1/DOPC <5 <5

Example 5

Groups of 5 mice each were immunized subcutaneously with 10 μg of HAantigen, alone or with increasing amounts ofN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide (Cmpd of Example 1)/DOPCas cited in Table 4. The mice were boosted with the same combinations 2weeks and 4 weeks following initial immunization. At 7 weeks postimmunization, the mice were bled and the HA-specific IgG2a titers weredetermined. This determination was performed by serial dilution of theserum samples by standard serum ELISA in HA-coated microtiter plates.IgG2a data is the serum dilution achieving the end point (2× baseline)and is the geometric mean for the 5 mice per group.

TABLE 4 HA Specific IgG2a, Serum Dilution End In Vivo Immunization GroupPoint Phosphate Buffered Saline (PBS) <50 HA 5.0E+03 HA + Cmpd ofExample 1 (1.0 MPK)/ DOPC 2.5E+05 HA + Cmpd of Example 1 (0.3 MPK)/DOPC1.3E+06 HA + Cmpd of Example 1 (0.1 MPK)/DOPC 1.1E+06 HA + Cmpd ofExample 1 (0.03 MPK)/DOPC 5.0E+05 HA + Cmpd of Example 1 (0.01 MPK)/DOPC2.5E+05

Example 6

Antigen dependent interferon-gamma (IFNgamma) responses were determinedin spleenocyte cultures established from the same animals for whichIgG2a antibody responses were determined in Example 5. The spleens fromthe animals were removed, combined to form two pools for each group of 5animals, minced to create single cell suspensions, and placed in culturein 96 well microtiter plates. Each pool generated three wells for acontrol PBS challenge and three wells for a 10 mg HA challenge. Thecultures were then incubated at 37° C. for 72 hours. The medium was thenremoved and the interferon-gamma generated was measured (pg/ml) by anELISA assay (Table 5). The IFNgamma data is reported as the geometricmean value for each pool using triplicate measurements.

TABLE 5 In Vitro Challenge of Isolated Spleenocytes, (IFNgamma, pg/ml)Control PBS HA Antigen In Vivo Immunization Group Challenge ChallengePhosphate Buffered Saline (PBS) 199.50 224.37 HA 189.74 236.64 HA + Cmpdof Example 1 (1.0 MPK)/DOPC 194.80 278.87 HA + Cmpd of Example 1 (0.3MPK)/DOPC 184.23 861.42 HA + Cmpd of Example 1 (0.1 MPK)/DOPC 189.74805.00 HA + Cmpd of Example 1 (0.03 MPK)/ 179.44 1219.23 DOPC HA + Cmpdof Example 1 (0.01 MPK)/ 204.82 1167.97 DOPC

Example 7

The ability of N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide (Cmpd of Example 1) toinduce tumor necrosis factor (TNF) production in human peripheralmononuclear cells (PBMC) was determined. The human peripheral bloodmononuclear cells were prepared from human volunteers and placed inculture in 96 well microtiter plates. The Cmpd of Example 1 was added tothe wells at the following concentrations: 30, 10, 3.3, 1.1, 0.37, 0.13,0.043, and 0.014 μM. The cells were then incubated overnight at 37° C.The medium was removed and TNF concentration (ng/mL) was measured byELISA assay (Table 6).

TABLE 6 Cmpd of Example 1 Concentration μM TNF ng/mL 0.014 0.13 0.0430.17 0.13 0.35 0.37 2.51 1.1 7.07 3.3 28.73 10 31.46 30 29.47

Example 8

The viral protection activity ofN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide(Cmpd of Example 1) was evaluated in Balb/c male mice (Charles River,Wilmington, Mass.) infected intranasally with mouse-adapted H1N1A/Puerto Rico/8/34 (obtained from American Type Culture Collection,Manassas, Va.). Four weeks prior to infection, groups of 10 mice eachwere immunized with 1. PBS; 2. 10 μg HA; or 3. 10 μg HA+0.1 mg/Kg ofCmpd of Example 1 in DOPC liposomes, respectively. Two weeks prior toinfection, the same groups were boosted with their correspondingimmunizing doses. Survival of mice was monitored for 11 days followingintranasal infection and the data is presented in Table 7 as percentsurvival on each day. One mouse from group 1, and two mice from group 2failed to achieve infection as determined from lack of weight losswithin the first 3 days of infection. Therefore, by day 5 group 1 wascomprised of 9 mice, group 2 was comprised of 8 mice, and groups 3 and 4were comprised of 10 mice, each.

TABLE 7 Immunization Group (Percent Survival) HA + Cmpd of Day PBS HAExample 1 1 100 100 100 2 100 100 100 3 100 100 100 4 100 100 100 5 100100 100 6 100 100 100 7 77.8 100 100 8 66.7 75.0 100 9 44.4 75.0 100 1011.1 50.0 100 11 0 50.0 100

Example 9

The immune activation activity ofN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide(Cmpd of Example 1) was evaluated in a mouse prophylactic anti-tumorimmunization model. Groups of C57/Bl male mice (Charles River,Wilmington, Mass.) were immunized and boosted twice at two weekintervals with 1) PBS; 2) 20 μg ovalbumin; or 3) 20 μg ovalbumin+1.0mg/Kg Cmpd of Example 1. One week following the final boost, each mousewas injected intradermally with 4E5 B16Ova melanoma tumor cells. Micewere sacrificed 11 days following tumor injection, tumors were measuredat their major and minor diameters, and the products of the twomeasurements were determined. The mean tumor size in mm²+/−standarddeviation (s.d.) for each group was determined. The results arepresented in Table 8.

TABLE 8 Immunization Number Mean Tumor Material of Mice Size (s.d.) PBS7 10.21 (4.34) Ovalbumin 8 10.18 (8.95) Ovalbumin + 8  0.99 (0.81) Cmpdof Example 1

Example 10

The dose sparing activity of N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide (Cmpd of Example 1) wasevaluated in mice immunized with varying amounts of HA with and withoutCmpd of Example 1. Groups of five Balb/c male mice (Charles River,Wilmington, Mass.) were immunized with 1 μg, 5 μg, or 15 μg of HA withor without 0.1 mg/kg of Cmpd of Example 1. The mice were then boostedwith the same preparations at 2 weeks and at 4 weeks post immunization.Three weeks following the final boost, the mice were bled and titers ofHA-specific IgG1 and IgG2a were determined by serial dilution of theserum samples using a standard serum ELISA assay in HA-coated microtiterplates. The IgG1 and IgG2a data is presented in Table 9 as the serumdilution that achieved the end point (2× baseline) and is the geometricmean for 5 mice per group. The addition of 0.1 mg/Kg of Cmpd of Example1 to HA greatly enhanced the antibody response to this antigen.

TABLE 9 Immunization Group IgG1 End Point IgG2a End Point HA 1 μg 2.5 E43.3 E2 HA 5 μg 6.7 E4 1.0 E3 HA 15 μg 6.7 E4 2.5 E3 HA 1 μg + Cmpd ofExample 1 1.7 E7 3.3 E6 HA 5 μg + Cmpd of Example 1 1.4 E7 2.5 E7 HA 15μg + Cmpd of Eaxmple 1 1.1 E7 1.0 E8

Example 11

The local in vivo activity of N-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl) octadecanamide (Cmpd of Example 1) wasevaluated in groups of four Balb/c male mice (Charles River) andcompared to the activity of resiquimod (a comparator compound).Solutions of the Cmpd of Example 1 or resiquimod were injectedsubcutaneously into four separate groups of mice for evaluation at thetime points of 1 hour, 3 hour, 6 hours, and 18 hours post dose. Thefinal dose for either compound was 1.0 mg/kg. At each time point, themice were bled, sacrificed, and the draining axial and brachial lymphnodes were removed and placed in RNA preservation fluid (RNAlaterreagent obtained from Ambion Corporation, Austin, Tex.). Serum sampleswere analyzed for TNF protein concentration (pg/ml) by ELISA as ameasure of systemic presence of this cytokine. The draining lymph nodeswere processed for measurement of TNF mRNA gene expression byquantitative PCR (7900HT Thermocycler obtained from Applied Biosy stems,Carlsbad, Calif.). The data reported (Table 10) is the mean+/−standarddeviation (s.d.) for each group. The “not detected” level for serum TNFconcentration was less than 10 pg/ml. The induction of TNF mRNA geneexpression in the draining lymph nodes without detection of TNF proteinin the serum after the injection of the Cmpd of Example 1 demonstratesthat the cytokine induction effects of the Cmpd of Example 1 areprimarily local.

TABLE 10 TNF mRNA Gene Expression in Lymph Nodes [fold increase versusSerum TNF [pg/ml (s.d.)] na 

 ve control (s.d.)] Time Cmpd of Cmpd of (hours) Example 1 ResiquimodExample 1 Resiquimod 1 not detected 4082 (873) 0.65 (0.04) 14.19 (3.83)3 not detected 107 (35) 1.62 (1.23) 4.82 (0.70) 6 not detected 18 (4)7.23 (2.07) 1.39 (0.27) 18 not detected not detected 1.55 (0.28) 0.90(0.23)

The present invention thus provides the compound of Formula I, as wellas pharmaceutical compositions and formulations thereof. In someembodiments, the compound of Formula I is incorporated into a liposomebased formulation. One may also incorporate an antigen admixed with oradministered separately but in combination with such formulation. Forexample, an antigen may be formulated within the lumen of theself-assembling liposome particle. Such liposomes would includecomposites of such substances in proportions best suited to yield stableparticles of desired sizes and diameters. Sizes can be of the sub micronrange to mimic viral pathogens and micron size to mimic bacterialantigens. These sizes can be controlled by particle composition andprocess of formation.

In some embodiments of the methods disclosed herein, the compound ofFormula I (e.g., in a pharmaceutical composition disclosed herein) isadministered to a localized tissue region, such as into a tumor mass. Insome of these embodiments, the compound of Formula I is administered tolocalized tissue, such as a tumor mass, in a liposome formulation. Acancer vaccine may also be included.

A “localized tissue region” will generally be a relatively small portionof the body, e.g., less than 10 percent by volume, and often less than 1percent by volume. For example, depending on the size of, e.g., a solidtumor or cancerous organ, the localized tissue region will typically beon the order of no more than about 500 cubic centimeters (cm³), oftenless than about 100 cm³, and in many instances 10 cm³ or less. For someapplications the localized tissue region will be 1 cm³ or less (e.g.,for small tumor nodules, viral lesions, or vaccination sites). However,in certain instances the localized tissue region may be a particularlylarge region, up to several liters, for example, to treat metastasizedcancer within the entire peritoneal cavity. The localized tissue regionmay be, for example, a cancer, a viral infected lesion, or organ, orvaccination site. It may be, for example, a solid tumor, lymph tissue,reticuloendothelial system, bone marrow, mucosal tissue, etc. Thelocalized tissue region may be, e.g., a breast cancer tumor, stomachcancer tumor, lung cancer tumor, head or neck cancer tumor, colorectalcancer tumor, renal cell carcinoma tumor, pancreatic cancer tumor, basalcell carcinoma tumor, cervical cancer tumor, melanoma cancer tumor,prostate cancer tumor, ovarian cancer tumor, or bladder cancer tumor.Delivery of the compound of Formula I to a localized tissue region maybe in conjunction with image guiding techniques using, for example,ultrasound, MRI, and real-time X-ray (fluoroscopy).

In some embodiments of the pharmaceutical compositions and methodsdisclosed herein, the pharmaceutical composition further comprises anantigen in an amount effective to generate an immune response againstthe antigen. In some embodiments, the antigen is a vaccine. Vaccinesinclude any material administered to raise either humoral and/or cellmediated immune response, such as live or attenuated viral and bacterialimmunogens and inactivated viral, tumor-derived, protozoal,organism-derived, fungal, and bacterial immunogens, toxoids, toxins,polysaccharides, proteins, glycoproteins, peptides, cellular vaccines(e.g., using dendritic cells), DNA vaccines, recombinant proteins,glycoproteins, and peptides. Exemplary vaccines include vaccines forcancer, BCG, cholera, plague, typhoid, hepatitis A, B, and C, influenzaA and B, parainfluenza, polio, rabies, measles, mumps, rubella, yellowfever, tetanus, diphtheria, hemophilus influenza b, tuberculosis,meningococcal and pneumococcal vaccines, adenovirus, HIV, chicken pox,cytomegalovirus, dengue, feline leukemia, fowl plague, HSV-1 and HSV-2,hog cholera, Japanese encephalitis, respiratory syncytial virus,rotavirus, papilloma virus, severe acute respiratory syndrome (SARS),anthrax, and yellow fever. See also, e.g., vaccines disclosed inInternational Publication No. WO 02/24225 (Thomsen et al.).

Antigens can be co-delivered with a compound of Formula I, for example,in admixture in a pharmaceutical composition according to the presentinvention. Such pharmaceutical compositions may include the compound inFormula I in liposomes. This may allow the compound of Formula I toreach, for example, antigen presenting cells at or around the same timeas the antigen. In other embodiments, the compound of Formula I and theantigen may be administered separately at or about the same time.Co-delivering a vaccine adjuvant (e.g., an IRM compound such as acompound of Formula I) and an antigen to an immune cell can increase theimmune response to the antigen and improve antigen-specificimmunological memory. Optimal delivery may occur, for example, when theadjuvant and the antigen are processed within an antigen presenting cellat the same time.

In addition to the delivery methods mentioned specifically above, acompound of Formula I (e.g., in a pharmaceutical composition disclosedherein) may be administered in any other suitable manner (e.g.,non-parenterally or parenterally). As used herein, non-parenterallyrefers to administration through the digestive tract, including by oralingestion. Parenterally refers to administration other than through thedigestive tract which would include nasal (e.g., transmucosally byinhalation), topical, ophthalmic, and buccal adminstration, but inpractice usually refers to injection (e.g., intravenous, intramuscular,subcutaneous, intratumoral, or transdermal) using, for example,conventional needle injection, injection using a microneedle array, orany other known method of injection.

The compound of Formula I may be provided in any pharmaceuticalcomposition suitable for administration to a subject and may be presentin the pharmaceutical composition in any suitable form (e.g., asolution, a suspension, an emulsion, or any form of mixture). Thepharmaceutical composition may be formulated with any pharmaceuticallyacceptable excipient, carrier, or vehicle. In some embodiments, thepharmaceutically acceptable carrier comprises water (e.g., phosphate orcitrate buffered saline). In some embodiments, the pharmaceuticallyacceptable carrier comprises an oil (e.g., corn, sesame, squalene,cottonseed, soybean, or safflower oil). The pharmaceutical compositionmay further include one or more additives including skin penetrationenhancers, colorants, fragrances, flavorings, moisturizers, thickeners,suspending agents, surfactants, and dispersing agents.

In addition to antigens specifically described above, the pharmaceuticalcompositions and methods of the present disclosure can include otheradditional active agents, e.g., in admixture or administered separately.Such additional agents can include a chemotherapeutic agent, acytotoxoid agent, an antibody, an antiviral agent, a cytokine, a tumornecrosis factor receptor (TNFR) agonist, or an additional immuneresponse modifier. TNFR agonists that may be delivered in conjunctionwith the compound of Formula I include CD40 receptor agonists, such asdisclosed in copending application U.S. Patent Publication 2004/0141950(Noelle et al.). Other active ingredients for use in combination with anIRM preparation of the present invention include those disclosed in,e.g., U.S. Patent Publication No. 2003/0139364 (Krieg et al.).

In some embodiments, a pharmaceutical composition according to thepresent invention may be a conventional topical dosage formulation(e.g., a cream, an ointment, an aerosol formulation, a non-aerosolspray, a gel, or a lotion). Suitable types of formulations aredescribed, for example, in U.S. Pat. No. 5,238,944 (Wick et al.); U.S.Pat. No. 5,939,090 (Beaurline et al.); U.S. Pat. No. 6,245,776(Skwierczynski et al.); European Patent No. EP 0394026 (Schultz); andU.S. Patent Publication No. 2003/0199538 (Skwierczynski et al.).

The compound of Formula I has been shown to induce the production ofTNF-α as described above. The ability to induce TNF production indicatesthat the compound of Formula I is useful as an immune response modifierthat can modulate the immune response in a number of different ways,rendering it useful in the treatment of a variety of disorders. Othercytokines whose production may be induced by the administration of thecompound of Formula I generally include Type I interferons (e.g.,INF-α), IL-1, IL-6, IL-8, IL-10, IL-12, MIP-1, MCP-1, and a variety ofother cytokines. Among other effects, these and other cytokines inhibitvirus production and tumor cell growth, making the compound of Formula Iuseful in the treatment of viral diseases and neoplastic diseases.Accordingly, the invention provides a method of inducing cytokinebiosynthesis in an animal comprising administering an effective amountof the compound of Formula I (e.g., in a pharmaceutical composition) tothe animal. The animal to which the compound of Formula I isadministered for induction of cytokine biosynthesis may have a disease(e.g., a viral or neoplastic disease), and administration of thecompound may provide therapeutic treatment. Also, the compound ofFormula I may be administered to the animal before the animal acquiresthe disease so that administration of the compound of Formula I mayprovide a prophylactic treatment.

In addition to the ability to induce the production of cytokines, thecompound of Formula I may affect other aspects of the innate immuneresponse. For example, natural killer cell activity may be stimulated,an effect that may be due to cytokine induction. IRM activity of thecompound of Formula I also may include activating macrophages, which inturn stimulate secretion of nitric oxide and the production ofadditional cytokines. IRM activity of the compound of Formula I also mayinclude inducing cytokine production by T cells, activating T cellsspecific to an antigen, and/or activating dendritic cells. Further, IRMactivity of the compound of Formula I may include proliferation anddifferentiation of B-lymphocytes. IRM activity of the compound ofFormula I also may affect the acquired immune response. For example, IRMactivity can include inducing the production of the T helper type 1(T_(H)1) cytokine IFN-γ and/or inhibiting the production of the T helpertype 2 (T_(H)2) cytokines IL-4, IL-5 and/or IL-13.

Exemplary conditions that may be treated by administering the compoundof Formula I include:

(a) viral diseases such as diseases resulting from infection by anadenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus(e.g., an orthopoxvirus such as variola or vaccinia, or molluscumcontagiosum), a picornavirus (e.g., rhinovirus or enterovirus), anorthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g.,parainfluenzavirus, mumps virus, measles virus, and respiratorysyncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g.,papillomaviruses, such as those that cause genital warts, common warts,or plantar warts), a hepadnavirus (e.g., hepatitis B virus), aflavivirus (e.g., hepatitis C virus or Dengue virus), or a retrovirus(e.g., a lentivirus such as HIV);

(b) bacterial diseases such as diseases resulting from infection bybacteria of, for example, the genus Escherichia, Enterobacter,Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter,Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus,Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus,Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia,Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, orBordetella;

(c) other infectious diseases such as chlamydia, fungal diseases (e.g.,candidiasis, aspergillosis, histoplasmosis, or cryptococcal meningitis),or parasitic diseases (e.g., malaria, pneumocystis carnii pneumonia,leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosomeinfection);

(d) neoplastic diseases such as intraepithelial neoplasias, cervicaldysplasia, actinic keratosis, basal cell carcinoma, squamous cellcarcinoma, renal cell carcinoma, Kaposi's sarcoma, melanoma, leukemias(e.g., myelogenous leukemia, chronic lymphocytic leukemia, multiplemyeloma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, B-celllymphoma, and hairy cell leukemia), breast cancer, lung cancer, prostatecancer, colon cancer, and other cancers;

(e) T_(H)2-mediated, atopic diseases such as atopic dermatitis oreczema, eosinophilia, asthma, allergy, allergic rhinitis, and Ommen'ssyndrome;

(f) certain autoimmune diseases such as systemic lupus erythematosus,essential thrombocythaemia, multiple sclerosis, discoid lupus, andalopecia areata; and

(g) diseases associated with wound repair such as inhibition of keloidformation and other types of scarring (e.g., enhancing wound healing,including chronic wounds).

The mechanism for the antiviral and antitumor activity of the compoundof Formula I may be due in substantial part to enhancement of the immuneresponse by induction of various important cytokines (e.g., at least oneof tumor necrosis factor, interferons, or interleukins). Such compoundshave been shown to stimulate a rapid release of certainmonocyte/macrophage-derived cytokines and are also capable ofstimulating B cells to secrete antibodies which play an important rolein these IRM compounds' antiviral and antitumor activities.

It will be understood that in the treatment of the diseases mentionedabove, for example, the compound of Formula I can be used in combinationwith other therapies such as the active agents mentioned above and otherprocedures (e.g., chemoablation, laser ablation, cryotherapy, andsurgical excision).

An amount of a compound effective to induce cytokine biosynthesis is anamount sufficient to cause one or more cell types, such as monocytes,macrophages, dendritic cells and B-cells to produce an amount of one ormore cytokines such as, for example, IFN-α, TNF-α, IL-1, IL-6, IL-lO andIL-12 that is increased over a background level of such cytokines. Theprecise amount will vary according to factors known in the art but isexpected to be a dose of about 100 nanograms per kilograms (ng/kg) toabout 50 milligrams per kilogram (mg/kg), in some embodiments about 10micrograms per kilogram (μg/kg) to about 5 mg/kg. The invention alsoprovides a method of treating a viral infection in an animal and amethod of treating a neoplastic disease in an animal comprisingadministering an effective amount of a compound or pharmaceuticalcomposition of the invention to the animal. An amount effective to treator inhibit a viral infection is an amount that will cause a reduction inone or more of the manifestations of viral infection, such as virallesions, viral load, rate of virus production, and mortality as comparedto untreated control animals. The precise amount that is effective forsuch treatment will vary according to factors known in the art but isexpected to be a dose of about 100 ng/kg to about 50 mg/kg, in someembodiments about 10 μg/kg to about 5 mg/kg. An amount of a compound orpharmaceutical composition effective to treat a neoplastic condition isan amount that will cause a reduction in tumor size or in the number oftumor foci. Again, the precise amount will vary according to factorsknown in the art but is expected to be a dose of about 100 ng/kg toabout 50 mg/kg, in some embodiments about 10 μg/kg to about 5 mg/kg. Themethods of the present invention may be performed on any suitablesubject. Suitable subjects include animals such as humans, non-humanprimates, rodents, dogs, cats, horses, pigs, sheep, goats, or cows.

The composition of a formulation suitable for practicing the invention,the precise amount of a compound of Formula I effective for methodsaccording to the present invention, and the dosing regimen, for example,will vary according to factors known in the art including the nature ofthe carrier, the state of the subject's immune system (e.g., suppressed,compromised, stimulated), the method of administering the compound ofFormula I, and the species to which the formulation is beingadministered. Accordingly, it is not practical to set forth generallythe composition of a formulation that includes a compound of Formula I,an amount of a compound of Formula I that constitutes an effectiveamount, or a dosing regimen that is effective for all possibleapplications. Those of ordinary skill in the art, however, can readilydetermine appropriate formulations, amounts of the compound of FormulaI, and dosing regimen with due consideration of such factors.

In some embodiments, the methods of the present invention includeadministering a compound of Formula I to a subject in a formulation, forexample, having a concentration of the compound from about 0.0001% toabout 20% (unless otherwise indicated, all percentages provided hereinare weight/weight with respect to the total formulation), although insome embodiments the compound of Formula I may be administered using aformulation that provides the compound in a concentration outside ofthis range. In some embodiments, the method includes administering to asubject a formulation that includes from about 0.01% to about 1% of thecompound of Formula I, for example, a formulation that includes about0.1% to about 0.5% compound of Formula I.

In some embodiments, the methods of the present invention includeadministering sufficient compound to provide a dose of, for example,from about 100 ng/kg to about 50 mg/kg to the subject, although in someembodiments the methods may be performed by administering compound in adose outside this range. In some of these embodiments, the methodincludes administering sufficient compound to provide a dose of fromabout 10 μg/kg to about 5 mg/kg to the subject, for example, a dose offrom about 100 μg/kg to about 1 mg/kg. In some embodiments, the methodsof the present invention may include administering sufficient compoundto provide a dose of, for example, from about 0.01 mg/m² to about 10mg/m². Alternatively, the dose may be calculated using actual bodyweight obtained just prior to the beginning of a treatment course. Forthe dosages calculated in this way, body surface area (m²) is calculatedprior to the beginning of the treatment course using the Dubois method:m²=(wt kg^(0.425)×height cm^(0.725))×0.007184.

In some embodiments of the methods disclosed herein, the compound ofFormula I may be administered, for example, from a single dose tomultiple doses per week, although in some embodiments the methods of thepresent invention may be performed by administering the compound ofFormula I at a frequency outside this range. In some embodiments, thecompound of Formula I may be administered from about once per month toabout five times per week. In some embodiments, the compound of FormulaI is administered once per week.

Since the compound of Formula I can be formulated to provide reducedsystemic levels of the compound while inducing a high levels ofcytokines, it is believed to be very useful for providing an enhancedlocal immune response while minimizing undesirable systemic sideeffects. This may be advantageous for many uses, such as directadministration to a tumor and/or as a vaccine adjuvant.

Objects and advantages of this invention are illustrated by the aboveexamples, but the particular materials and amounts thereof recited, aswell as other conditions and details, should not be construed to undulylimit this invention.

The complete disclosures of the patents, patent documents, andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousmodifications and alterations to this invention will become apparent tothose skilled in the art without departing from the scope and spirit ofthis invention. It should be understood that this invention is notintended to be unduly limited by the illustrative embodiments andexamples set forth herein and that such examples and embodiments arepresented by way of example only with the scope of the inventionintended to be limited only by the claims set forth herein as follows.

What is claimed is:
 1. A method of reducing tumor size by administeringthe compoundN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide,or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the compound is administered directly into a localized tumormass.
 3. The method of claim 2, wherein the tumor size is reduced by 30to 100%.
 4. The method of claim 3, wherein the tumor size is reduced by50 to 90%.
 5. The method of claim 4, wherein the tumor size is reducedby 60 to 80%.
 6. The method of claim 5, wherein the tumor size isreduced by 70%.
 7. The method of claim 1, wherein the tumor size isreduced by at least 90%.
 8. A method of inducing an immune response in alocalized tissue region by administering the compoundN-(4-{[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]oxy}butyl)octadecanamide,or a pharmaceutically acceptable salt thereof.
 9. The method of claim 8,wherein the compound is administered directly into a localized tumormass.